CN107812182B - The application of neuraminidase and its inhibitor in the preparation of drugs for inhibiting hepatic gluconeogenesis - Google Patents

Abstract

The invention discloses application of neuraminidase and an inhibitor in preparation of a medicine for inhibiting hepatic gluconeogenesis. Zanamivir and oseltamivir phosphate are known to be effective inhibitors of neuraminidase, the inhibition effect of coptisine on neuraminidase is also disclosed in the previous patent application of the applicant, and the applicant finds that salvianolic acid B can inhibit the activity of neuraminidase in vitro and is an inhibitor of neuraminidase; in vivo experiments prove that the neuraminidase inhibitors can effectively inhibit the neuraminidase activity in the liver, and further inhibit the gluconeogenesis of the liver. In conclusion, neuraminidase and inhibitors thereof can be used for preparing medicines for inhibiting hepatic gluconeogenesis, and can be used for treating diabetes, obesity and non-alcoholic fatty liver.

Description

The application of neuraminidase and its inhibitor in the preparation of drugs for inhibiting hepatic gluconeogenesis

technical field

The invention belongs to the field of biomedicine and relates to the application of enzymes and enzyme inhibitors, in particular to the application of neuraminidase and its inhibitors in the preparation of medicines for inhibiting hepatic gluconeogenesis.

Background technique

Neuraminidine is a class of natural sugar acid compounds that are widely present in living organisms. Now it is determined that there are more than 50 natural derivatives of neuraminic acid, among which N-acetylneuraminic acid and N-glycolylneuraminic acid are the more common ones. Neuraminidine is usually linked to the end of glycoconjugates such as glycoproteins and glycolipids in the form of short chain residues. Neuraminidine is an important biological information transmission molecule, and the neuraminidation modification of cell surface glycoproteins and glycolipids plays a crucial role in many biological processes, including cell adhesion, antigen recognition, and signal transduction.

In vivo, neuraminidase is an enzyme related to neuraminic acid, which plays an indispensable role in many important physiological processes in vivo. Neuraminidase can cleave the glycosidic bond connecting the end of the glycoconjugate to the neuraminic acid residue, thereby hydrolyzing the neuraminic acid glycoconjugate and freeing the neuraminic acid.

Neuraminidase inhibitors are a class of substances that inhibit the activity of neuraminidase, such as zanamivir and oseltamivir phosphate. Zanamivir and oseltamivir phosphate are commonly used clinical drugs for the treatment of influenza. Influenza virus binds to neuraminic acid residues on the surface of host cells through hemagglutinin on its surface, and then invades host cells. Subsequently, its genes are replicated and expressed in host cells to form new influenza viruses. In the release stage of the new virus, neuraminidase catalyzes the hydrolysis of the neuraminic acid glycosidic bond between the surface hemagglutinin of the mature influenza virus and the neuraminic acid residues of the host cell, so that the new virus particles are separated from the infected cells and further Spread in the host. Zanamivir and oseltamivir phosphate inhibit the separation of new virus particles from infected cells by inhibiting the activity of neuraminidase, and inhibit their spread in the host to achieve therapeutic purposes.

On March 22, 2016, the applicant submitted a patent application with the application number of "201610166253.5" and the invention titled "Application of Neuraminidase and its Inhibitors in Myocardial Ischemia and Myocardial Infarction", which discloses neural The application of neuraminidase and its inhibitors in myocardial ischemia and myocardial infarction provides the correlation between neuraminidase and myocardial ischemia injury, and proves that myocardial ischemic injury can be alleviated by inhibiting the activity of neuraminidase. Aminidase can be used as a target for screening drugs that prevent, alleviate and/or treat myocardial ischemia injury; this application also demonstrates the alleviation effect of neuraminidase inhibitors on myocardial ischemia injury, and neuraminidase inhibitors can reduce nerve Levels of aminidase ameliorate myocardial ischemic injury.

The applicant also found that neuraminidase is related to a variety of diseases in subsequent studies, and after searching, it was found that the prior art did not disclose the relationship between neuraminidase and these diseases.

SUMMARY OF THE INVENTION

The purpose of the present invention is to provide the application of neuraminidase and its inhibitor in the preparation of medicines for inhibiting hepatic gluconeogenesis.

The above-mentioned purpose of the present invention is achieved through the following technical solutions:

Application of neuraminidase in the preparation of drugs for inhibiting hepatic gluconeogenesis.

The application of neuraminidase in the preparation of medicine for treating diabetes.

The application of neuraminidase in the preparation of medicaments for treating obesity.

Application of neuraminidase in the preparation of medicines for treating non-alcoholic fatty liver disease.

Application of a neuraminidase inhibitor in the preparation of a drug for inhibiting hepatic gluconeogenesis.

Application of a neuraminidase inhibitor in the preparation of a medicament for treating obesity.

Application of a neuraminidase inhibitor in the preparation of a medicament for treating obesity.

The application of a neuraminidase inhibitor in the preparation of a medicament for treating non-alcoholic fatty liver disease.

Use of coptisine or salvianolic acid B and pharmaceutically acceptable salts thereof for preparing a neuraminidase inhibitor.

Use of zanamivir, oseltamivir phosphate, coptisine or salvianolic acid B and pharmaceutically acceptable salts thereof in the preparation of medicines for inhibiting hepatic gluconeogenesis, treating diabetes, treating obesity or treating non-alcoholic fatty liver disease .

It is known that zanamivir and oseltamivir phosphate are effective inhibitors of neuraminidase, and the inhibitory effect of coptisine on neuraminidase has also been disclosed in the applicant's previous patent application, and the applicant has found that salvianol Acid B can inhibit the activity of neuraminidase in vitro and is an inhibitor of neuraminidase; in vivo experiments have shown that these neuraminidase inhibitors can effectively inhibit the activity of neuraminidase in the liver, thereby inhibiting hepatic glucose Alien. In conclusion, neuraminidase and its inhibitors can be used to prepare drugs for inhibiting hepatic gluconeogenesis for the treatment of diabetes, obesity and non-alcoholic fatty liver disease.

Description of drawings

Figure 1 is the liver gluconeogenesis rate (%) of mice in each group;

Figure 2 shows the neuraminidase activity (U/μL) of the mice in each group.

Detailed ways

The substantive content of the present invention is specifically described below with reference to the examples, but the protection scope of the present invention is not limited by this.

Example 1:

Fifty Kunming mice were randomly divided into 5 groups according to body weight and gender: control group and zanamivir, oseltamivir phosphate, coptisine, and salvianolic acid B administration group. The mice in the administration group were given 0.2mg/kg/d zanamivir (i.v.), 5mg/kg/d oseltamivir phosphate (p.o.), 40mg/kg/d coptisine (p.o.), 40mg/kg /d salvianolic acid B (p.o.), the control group was given an equal volume of vehicle 0.5% CMC-Na by intragastric administration for 4 consecutive weeks. Seven animals in each group were used to determine the gluconeogenesis rate. The specific method was as follows: after fasting for 12 hours after the last gavage, blood was collected from the tail vein to measure the fasting blood glucose, which was recorded as the blood glucose value at 0 min; L-a-alanine was injected, and blood was collected from the eyeball after 1 h, and the blood glucose level was measured at 60 min. The gluconeogenesis rate (%) was calculated according to the blood glucose values at 0 min and 60 min. Finally, the livers were sacrificed, and the liver glycogen was measured by anthrone-sulfuric acid method.

Results After 1 hour of intraperitoneal injection of glycogenic amino acids, blood glucose in each group increased, but the increase in blood glucose in the control group was significantly greater than that in each administration group. The gluconeogenesis rate (%) was calculated according to the blood glucose values at 0 min and 60 min in each group, as follows Table and Figure 1 show:

group Hepatic gluconeogenesis rate (%) Oseltamivir phosphate administration group 24.88±3.55 control group 34.72±3.64 Coptis administration group 27.96±3.82 Zanamivir administration group 26.55±3.73 Salvianolic acid B administration group 24.57±3.68

The results of liver glycogen determination showed that the average content of liver glycogen in the control group was 0.55g/100g, and the average content of liver glycogen in the zanamivir, oseltamivir phosphate, coptisine, and salvianolic acid B administration groups was all 0.7g. /100g or more, there was significant difference with the control group (P<0.05).

The remaining 3 animals in each group were used to measure the activity of neuraminidase in the liver. The specific method was as follows: 2 hours after the last gavage, they were killed by decapitation, and the liver was quickly taken out to measure the activity of neuraminidase in the liver. The specific method is as follows: the mice were killed by decapitation, the liver was quickly taken out, placed on ice, rinsed with ice-cold physiological saline, dried with filter paper, weighed, and added with 0.25mmol/L sucrose at a ratio of 500g/L Homogenize with 1mmol/L EDTA homogenate, fully homogenize the homogenate, centrifuge the homogenate at 2000g × 10min, further centrifuge the obtained supernatant at 78000g for 90min, take the supernatant, use ELISA method to detect rat liver neuraminic acid For the enzyme level, the detection kit was a neuraminidase detection kit (purchased from Biyuntian), which was determined according to the operating instructions of the detection kit. The measurement results are shown in the following table and Figure 2:

group Neuraminidase activity (U/μL) Oseltamivir phosphate administration group 15.13±1.52 control group 23.55±1.84 Coptis administration group 17.32±1.63 Zanamivir administration group 16.21±1.46 Salvianolic acid B administration group 15.29±1.77

The above examples prove that zanamivir, oseltamivir phosphate, coptisine, and salvianolic acid B can inhibit the activity of neuraminidase in vivo, thereby inhibiting hepatic gluconeogenesis by inhibiting the activity of neuraminidase in the liver. . Those skilled in the art know that inhibiting hepatic gluconeogenesis can treat obesity, non-alcoholic fatty liver disease, diabetes and other diseases.

Example 2:

A commercially available neuraminidase inhibitor screening kit P0309 (Beyotime, Beyotime) was used to test the inhibitory activity of salvianolic acid B in vitro, and the positive control drug was oseltamivir phosphate. Add 70 μL of buffer and 10 μL of neuraminidase solution to each well of the 96-well plate, then add 10 μL of different concentrations of the solution to be tested, shake and mix, incubate at 37°C for 5 min, add 10 μL of the solution containing the fluorescent substrate, and shake to mix. After 30min incubation at 37°C, fluorescence measurement was performed, wherein the excitation wavelength was 322nm and the emission wavelength was 450nm. According to the fluorescence readings, the inhibition rates of different test solutions were calculated, and the IC50 values of the positive control drugs oseltamivir phosphate and salvianolic acid B were further obtained. The results are shown in the table below.

Oseltamivir Phosphate Salvianolic acid B IC50 value (nmol/L) 13.5 195.5

The above examples prove that salvianolic acid B can inhibit the activity of neuraminidase in vitro, showing moderate neuraminidase inhibitory activity, and is an inhibitor of neuraminidase.

It is known that zanamivir and oseltamivir phosphate are effective inhibitors of neuraminidase, and the inhibitory effect of coptisine on neuraminidase has also been disclosed in the applicant's previous patent application, and the applicant has found that salvianol Acid B can inhibit the activity of neuraminidase in vitro and is an inhibitor of neuraminidase; in vivo experiments have shown that these neuraminidase inhibitors can effectively inhibit the activity of neuraminidase in the liver, thereby inhibiting hepatic glucose Alien. In conclusion, neuraminidase and its inhibitors can be used to prepare drugs for inhibiting hepatic gluconeogenesis for the treatment of diabetes, obesity and non-alcoholic fatty liver disease.

The function of the above embodiments is to specifically introduce the essential content of the present invention, but those skilled in the art should know that the protection scope of the present invention should not be limited to the specific embodiments.

Claims (1)

1. Use of a neuraminidase inhibitor, which is zanamivir or oseltamivir phosphate, for the preparation of a medicament for inhibiting hepatic gluconeogenesis, treating diabetes, treating obesity or treating non-alcoholic fatty liver disease.

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